Online Resource 1 The final concentrations of the applied technical enzymes in the matrices wheat flour, bread and cookies

-Amylase from Aspergillus oryzae [ppm] / Xylanase from Thermomyces lanuginosus [ppm] / Lipase from Thermomyces lanuginosus [ppm]
Flour 0 / 0.00 / 0.00 / 0.00
Flour 1 / 2.58 / 1.04 / 0.36
Flour 2 / 9.03 / 3.64 / 1.26
Flour 3 / 19.35 / 7.80 / 2.70
Flour 4 / 38.70 / 15.60 / 5.40
Bread 0 / 0.00 / 0.00 / 0
Bread 1 / 1.94 / 0.78 / 0.27
Bread 2 / 7.61 / 3.07 / 1.06
Bread 3 / 19.22 / 7.75 / 2.68
Bread 4 / 38.83 / 15.65 / 5.42
Cookie 0 / 0.00 / 0.00 / 0
Cookie 1 / 2.58 / 1.04 / 0.36
Cookie 2 / 9.03 / 3.64 / 1.26
Cookie 3 / 19.35 / 7.80 / 2.7
Cookie 4 / 38.70 / 15.60 / 5.4

Online Resource 2 The recipe for bread making

Component / Weight [g]
Rest-Wheat flour / 1381
Peanut fat / 68
Sugar / 27
Salt (NaCl) / 41
Yeast / 68
Pre-Mix enzyme / 52
Water / 889
Total / 2500

Online Resource 3 The recipe for cookies

Component / Weight [g]
Rest-Wheat flour / 255
Sunflower margarine / 100
Sugar / 100
Baking powder / 9
Pre-Mix enzyme / 36
Water / 100
Total / 600

Online Resource 4 Preparation of calibration standards for HPLC-MS/MS

The peptides were obtained from peptides&elephants GmbH (Potsdam, Germany).

Solvents

  • Extraction buffer: Dissolve 7.9g Ammonium bicarbonate, 240 g Urea, 0.78 g 1,4-Dithiothreitol in 1000 ml distilled water. All chemicals were from Carl Roth GmbH + Co. KG (Karlsruhe, Germany).
  • Buffer A: 100ml distilled water + 100ml Acetonitrile (Merck KGaA, Darmstadt, Germany) + 0.2 ml formic acid (Carl Roth GmbH + Co. KG, Karlsruhe, Germany).
  • Buffer B: 200 ml distilled water + 0.2 ml formic acid (Carl Roth GmbH + Co. KG, Karlsruhe, Germany).

Protein standard for digestion control

A stock solution of α-Amylase from Bacillus licheniformis was prepared by dissolving 5.46 mg in 1 ml of the extraction buffer.

ISTD for α-Amylase from Bacillus licheniformis

A corresponding specific peptide (peptide sequence: EGDSSVANSGLAALITDGPGGAK, MW = 2086.02) for α-Amylase from Bacillus licheniformis was prepared by dissolving 2.4 mg in 1 ml buffer A to get a stock solution. 416.7 µl were than diluted to 10 ml with buffer B to give DL1 ISTD solution. This (2 ml DL1 ISTD) was further diluted with buffer B (8 ml) to produce DL2 ISTD.

Peptide standard for isotope marked peptides

Stock solutions

Peptide / Peptide sequence / MW [g/mol] / Weight [mg] / Volume
buffer A [µl] / Concentration Stock solution SL [mg/ml]
Xylanase 493 heavy / GWNPGLNAR / 995.48 / 2.8 / 500 / 5600
Xylanase 599 heavy / TSGTVQTGCH
FDAWAR / 1749.18 / 2.3 / 500 / 4600
Amylase 329 heavy / ALSSALHER / 994.40 / 3.9 / 500 / 7800
Amylase 411 heavy / NWPIYK / 829.90 / 2.7 / 500 / 5400
Lipase 465 heavy / SGTLVPVTR / 941.07 / 2.0 / 500 / 4000
Lipase 575 heavy / GHDGFTSSWR / 1160.52 / 2.0 / 500 / 4000
Lipase 583 heavy / ITHTNDIVPR / 1177.40 / 3.1 / 500 / 6200

Dilutions

Peptide / Volume SL [µl] / DL 1 Isopepmix [100 ppm] / DL 2 Isopepmix [5 ppm]
Xylanase 493 heavy / 178.6 / Fill up with buffer B to 10 ml
DL1 – Isopepmix enzymes / Fill up 500 µl DL 1 with buffer B to 10 ml
DL2 – Isopepmix enzymes
Xylanase 599 heavy / 217.4
Amylase 329 heavy / 128.2
Amylase 411 heavy / 185.2
Lipase 465 heavy / 250.0 / Fill up with buffer B to 10 ml
DL1 – Isopepmix lipase / Fill up 500 µl DL 1 with buffer B to 10 ml
DL2 – Isopepmix lipase
Lipase 575 heavy / 250.0
Lipase 583 heavy / 161.3

Peptide standard for unmarked peptides

Stock solutions

Peptide / Peptide sequence / MW [g/mol] / Weight [mg] / Volume
buffer A [µl] / Concentration Stock solution SL [mg/ml]
Xylanase 493 / GWNPGLNAR / 985.41 / 2.5 / 500 / 5000
Amylase 411 / NWPIYK / 821.37 / 2.2 / 500 / 4400
Lipase 465 / SGTLVPVTR / 930.61 / 2.0 / 500 / 4000
Lipase 575 / GHDGFTSSWR / 1150.33 / 2.2 / 500 / 4400
Lipase 583 / ITHTNDIVPR / 1167.51 / 2.5 / 500 / 5000

Dilutions

Peptide / Volume SL [µl] / DL 1 Isopepmix [100 ppm] / DL 2 Isopepmix [0.5 ppm]
Xylanase 493 / 200.0 / Fill up with buffer B to 10 ml
DL1 – Pepmix enzymes / Fill up 50 µl DL 1 with buffer B to 10 ml
DL2 – Pepmix enzymes
Amylase 411 / 227.3
Lipase 465 / 250.0 / Fill up with buffer B to 10 ml
DL1 – Pepmix lipase / Fill up 50 µl DL 1 with buffer B to 10 ml
DL2 – Pepmix lipase
Lipase 575 / 227.3
Lipase 583 / 200.0

Finally, a further dilution (DL3) was prepared by taking 2 ml DL2 – Pepmix enzymes/lipase and filling up with buffer B to 10 ml [0.1 ppm]

All the prepared solutions stored in brown bottles at -18°C.

Calibration

Calibration point (CP) / Volume DL3 Pepmix
Enzymes/lipase [µl] / Volume DL2 Isopepmix enzymes/lipase [µl] / Volume DL2 ISTD [µl] / Volume buffer B [µl] / Final concentration for CP [ppm]
0 / 2 / 40 / 100 / 858 / 0.0002
1 / 5 / 40 / 100 / 855 / 0.0005
2 / 10 / 40 / 100 / 850 / 0.0010
3 / 20 / 40 / 100 / 840 / 0.0020
4 / 100 / 40 / 100 / 760 / 0.0100
5 / 200 / 40 / 100 / 660 / 0.0200
6 / 400 / 40 / 100 / 460 / 0.0400
7 / 600 / 40 / 100 / 260 / 0.0600

Final concentration for the isotope marked peptidesis 0.2 ppm.

Final concentration of the specific peptide (EGDSSVANSGLAALITDGPGGAK) for α-Amylase from Bacillus licheniformisis 2.0 ppm

Online Resource 5A SDS-PAGE of Lipase FE-01 – the marked protein band was excised, treated and digested by trypsin and analysed by MALDI-TOF-MS as described in methods section.

Online Resource 5B Peptide calibration standard II for MALDI-TOF-MS

Peptide / [M+H]+ Mono isotopic / [M+H]+ Average
Bradykinin 1-7 / 757.3992 / 757.86
Angiotensin II / 1046.5418 / 1047.19
Angiotensin I / 1296.6848 / 1297.49
Substance P / 1347.7354 / 1348.64
Bombesin / 1619.8223 / 1620.86
Renin Substrate / 1758.9326 / 1760.03
ACTH clip 1-17 / 2093.0862 / 2094.43
ACTH clip 18-39 / 2465.1983 / 2466.68
Somatostatin 28 / 3147.4710 / 3149.57

Online Resource 6 Peptides identified (black) in sequence of lipase of Thermomyces lanuginosus by MALDI-TOF-MS

Sample 1

Sample 2

Sample 3

Online Resource 7 Results of BLAST and Align analysis for the sequence of lipase of Thermomyces lanuginosus (LIP_THELA) as determined by MALDI-TOF-MS analysis

The Basic Local Alignment Search Tool (BLAST) was applied to find regions of local similarity between sequences, which can be used to infer functional and evolutionary relationships between sequences as well as help identify members of gene families. The tool is available online at and the data was re-checked in March 2015 (original report is available). The selection data is summarized and limited to the first few hits.

Since the producer of the tested Lipase FE-0 gave the originating organism as from Aspergillus oryzae, an alignment was conducted of the sequence of Lipase from Thermomyces lanuginosus (LIP_THELA) with the corresponding hits from Aspergillus oryzae (G9M5R3_ASPNG and I7ZZJ6_ASPO3) with higher identity similarity (ca. 51 %). The sequence alignment was reproduced using the data in FASTA form available on analysis with the 'Align' tool available at The reproduction was achieved using the software Jalview (The Barton Group, University of Dundee, Scotland, UK), Vers 2.8. The peptides used for quantification/qualification with HPLC MS/MS analysis are bordered in red.

Code for identity:

Online Resource 8 Sequence alignment of amylases from three relevant sources ‐Aspergillus shirousami (AMY_ASPSH), Triticum aestivum (AMY3_WHEAT) and Bacillus licheniformis (AMY_BACLI)

Alignment was conducted online at and the FASTA files were evaluated using the software Jalview Vers.: 2.8 (The Barton group, University of Dundee, Scotland, UK). The peptides marked by the red/green frame were those applied for identification purposes.

Code for identity:

Online Resource 9 Sequence alignment of xylanases from the two relevant sources ‐Thermomyces lanuginosus (XYNA_THELA) and Triticum aestivum (AMY3_WHEAT)

Alignment was conducted online at and the FASTA files were evaluated using the software Jalview Vers.: 2.8 (The Barton group, University of Dundee, Scotland, UK). The peptides marked by the red frame were those applied for identification purposes.

Code for identity:

Online Resource 10 Identified peptides of -amylase in test material bread sample 3 (19 ppm -amylase from Aspergillus sp) and that of the internal standard peptide (ISTD 496) of the amylase from Bacillus licheniformis (AMY_BACLI).

Online Resource 11 HPLC-MS/MS analysis for peptides of -amylase in test material bread blank – the results show the absence of the corresponding identified peptides of -amylase from Aspergillus sp). The internal standard peptide (ISTD 496) of the amylase from Bacillus licheniformis (AMY_BACLI) is also shown in the last chromatogram.

Online Resource 12 Calculation of the recovery based on the internal standard peptide (ISTD 496) of the amylase from Bacillus licheniformis (AMY_BACLI) – example shows illustrates the recovery determination for lipase.

The concentration of the lipase 465 found in the digest obtained from wheat flour and baked goods can be determined from the above calibration. The value thus obtained is multiplied by an individual protein-peptide factor, which represents the mass ratio of the enzyme lipase to the lipase 465 (34.18). Since the amount administered in the wheat flour or baked goods is known (supporting information figure S1), the recovery can be calculated in [%]. The protein-peptide factors for -amylase (corresponding peptide -amylase 411) was 66.72 and for xylanase (corresponding peptide xylanase 493) 24.76 respectively.

Online Resource 13 Calculation of the recovery based on the isotope marked internal peptide standards for the individual enzymes – example shows illustrates the recovery determination for -amylase in wheat flour, dough, bread and cookies (overnight digestion).

Calibration curve: Area ratio = concentration of the amylase 411 / concentration of the isotope labelled amylase 411

Recovery was calculated similar to the method applied in supporting information figure S12.

Online Resource 14 Calculation of the recovery based on the isotope marked internal peptide standards for the individual enzymes – example shows illustrates the recovery determination for xylanase in wheat flour, dough, bread and cookies (overnight digestion).

Calibration curve: Area ratio = concentration of the xylanase 493 / concentration of the isotope labelled xylanase 493

Recovery was calculated similar to the method applied in supporting information figure S12.

Online Resource 15 Calculation of the recovery based on the isotope marked internal peptide standards for the individual enzymes – example shows illustrates the recovery determination forlipase in wheat bread and cookies (overnight digestion).

Calibration curve: Area ratio = concentration of the lipase 465 / concentration of the isotope labelled lipase 465

Recovery was calculated similar to the method applied in supporting information figure S12. White columns < LOD = level of detection; grey columns < LOQ = Level of quantification.

Online Resource 16 Changes in bread quality by using moderate amount of technical enzymes. The figures shows bread 0 – bread 4 (left to right) with different amounts of the enzymes as given in the supporting information figure S1.